Enzyme-Linked Immunosorbent Assay (ELISA)

Background

Enzyme-linked immunosorbent assay (ELISA) is a commonly used protein analysis method based on immunological reaction, which can be used to detect and quantify antibodies, antigens, proteins, glycoproteins, etc. ELISA is based on the immobilization and the enzyme labeling of antigen/antibody. Both immobilized and enzyme-labeled antigens or antibodies can retain their immunological activity, while enzyme-labeled antigens or antibodies also have enzyme activity. The main method of the ELISA is to coat a known antigen or antibody on a multi-well plate, and the test specimen reacts with the antigen or antibody on the multi-well plate. Then the enzyme-labeled antigen or antibody is added, which are then bound to the multi-well plate through the reaction. The amount of enzyme on the multi-well plate is proportional to the amount of test substance in the specimen. After adding the substrate of the enzyme reaction, the substrate is catalyzed by the enzyme to become a color-developing product. The amount of these products is directly related to the amount of the test substance in the specimen, so qualitative or quantitative analysis can be carried out according to the degree of color.

Types of ELISA

Direct ELISA

Enzyme-Linked Immunosorbent Assay (ELISA)

Coating the antigen on the multi-well plate, and then directly detect the antigen with enzyme-labeled antibody.
Indirect ELISA

Enzyme-Linked Immunosorbent Assay (ELISA)

First coat the known antigen on the multi-well plate, then add the sample to be tested to specifically bind to the antigen. After washing, add enzyme-labeled antibody, and the substrate is used for color development.
Sandwich ELISA

Enzyme-Linked Immunosorbent Assay (ELISA)

Sandwich ELISA is mostly used to detect antigens. Firstly, the known antibody is coated on the multi-well plate, and then the sample to be tested is added to specifically bind to the antibody. After washing, the antibody that binds to different epitopes of the antigen is added for detection and the substrate is used for color development.
Competitive ELISA

Enzyme-Linked Immunosorbent Assay (ELISA)

Competitive ELISA is to determine the content of the target analyte in the sample by quantifying the interference of the analyte on the reference antigen (or antibody) signal. Take the determination of antigen as an example. Firstly, the reference antigen is pre-coated on the multi-well plate. The antigen in the sample and the reference antigen are competitively combined with the enzyme-labeled antibody. After washing, perform the color test, and the color development result is inversely proportional to the concentration of the antigen in the sample.

Sandwich ELISA Procedure

  1. Dilute the specific antibody globulin to the optimum concentration (1-10μg/mL) with coating buffer, add 100 µL per well, overnight at 4℃, or water bath at 37℃ for 3 hours, then remove the coating solution and wash each well 3 times with washing buffer.
  2. Add the standard working solution to the first two rows of wells in turn, add two wells of each concentration of working solution in parallel, each with 100 µL. Add the sample to other wells, 100 µL per well (if the sample concentration is higher than the detection range, dilute it with standard and sample diluent for sampling). Cover the ELISA plate with membrane and incubate at 37°C for 90 minutes.
  3. Remove the liquid in the wells. Add 100 µL of the biotinylated antibody/antigen working solution to each well, mix well, cover the ELISA plate with membrane, and incubate at 37°C for 60 minutes.
  4. Remove the liquid in the wells, add 350 µL of washing solution to each well, soak for 1-2 minutes, aspirate the liquid in the microtiter plate, and pat dry gently on absorbent paper. Repeat this washing step 3 times.
  5. Add 100 µL of enzyme conjugate working solution to each well, mix well, cover the ELISA plate with membrane, and incubate at 37°C for 30 minutes.
  6. Remove the liquid in the wells, wash the plate 5 times, the method is the same as step 4.
  7. Add 90 µL of substrate solution to each well, mix well, cover with membrane, and incubate at 37°C in the dark for about 15 minutes (the specific incubation time should be handled according to the actual color development, not more than 30 minutes). When an obvious gradient appears in the standard well, perform the stop reaction.
  8. Add 50 µL of stop solution to each well to stop the reaction. The order of adding the stop solution should be the same as that of the substrate solution.
  9. Measure the optical density (OD value) of each well at 450 nm with a microplate reader.

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